Morris A J, Davenport R C, Tolan D R
Biology Department, Boston University, MA 02215, USA.
Protein Eng. 1996 Jan;9(1):61-7. doi: 10.1093/protein/9.1.61.
Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate): D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13] was changed to arginine by site-directed mutagenesis. The kcat of the resulting mutant protein, K146R, was 500 times slower than wild-type in steady-state kinetic assays for both cleavage and condensation of fructose-1,6-bis(phosphate), while the K(m) for this substrate was unchanged. Analysis of the rate of formation of catalytic intermediates showed K146R was significantly different from the wild-type enzyme and other enzymes mutated at this site. Single-turnover experiments using acid precipitation to trap the Schiff base intermediate on the wild-type enzyme failed to show a build-up of this intermediate on K146R. However, K146R retained the ability to form the Schiff base intermediate as shown by the significant amounts of Schiff base intermediate trapped with NaBH4. In the single-turnover experiments it appeared that the Schiff base intermediate was converted to products more rapidly than it was produced. This suggested a maximal rate of Schiff base formation of 0.022 s-1, which was close to the value of kcat for this enzyme. This observation is strikingly different from the wild-type enzyme in which Schiff base formation is > 100 times faster than kcat. For K146R it appears that steps up to and including Schiff base formation are rate limiting for the catalytic reaction. The carbanion intermediate derived from either substrate or product, and the equilibrium concentrations of covalent enzyme-substrate intermediates, were much lower on K146R than on the wild-type enzyme. The greater bulk of the guanidino moiety may destabilize the covalent enzyme-substrate intermediates, thereby slowing the rate of Schiff base formation such that it becomes rate limiting. The K146R mutant enzyme is significantly more active than other enzymes mutated at this site, perhaps because it maintains a positively charged group at an essential position in the active site or perhaps the Arg functionally substitutes as a general acid/base catalyst in both Schiff base formation and in subsequent abstraction of the C4-hydroxyl proton.
通过定点诱变将兔醛缩酶A [D-果糖-1,6-二磷酸：D-甘油醛-3-磷酸裂解酶，EC 4.1.2.13]的赖氨酸146位点突变为精氨酸。在果糖-1,6-二磷酸裂解和缩合的稳态动力学测定中，所得突变蛋白K146R的催化常数（kcat）比野生型慢500倍，而该底物的米氏常数（Km）未发生变化。对催化中间体形成速率的分析表明，K146R与野生型酶以及在此位点发生突变的其他酶存在显著差异。使用酸沉淀捕获野生型酶上席夫碱中间体的单周转实验未能显示K146R上该中间体的积累。然而，如用硼氢化钠捕获到大量席夫碱中间体所示，K146R保留了形成席夫碱中间体的能力。在单周转实验中，似乎席夫碱中间体转化为产物的速度比其生成速度更快。这表明席夫碱形成的最大速率为0.022 s-1，这与该酶的催化常数相近。这一观察结果与野生型酶截然不同，在野生型酶中席夫碱形成的速度比催化常数快100倍以上。对于K-146R而言，直至包括席夫碱形成在内的步骤似乎是催化反应的限速步骤。源自底物或产物的碳负离子中间体以及共价酶-底物中间体的平衡浓度，在K146R上比在野生型酶上低得多。胍基部分较大的体积可能会使共价酶-底物中间体不稳定，从而减缓席夫碱形成的速率，使其成为限速步骤。K146R突变酶比在此位点发生突变的其他酶活性显著更高，这可能是因为它在活性位点的关键位置保持了一个带正电荷的基团，或者精氨酸在席夫碱形成以及随后C4-羟基质子的夺取过程中作为一般酸碱催化剂发挥了功能替代作用。
